In vivo CRISPR-Cas9-mediated DNA chop identifies a cochlear outer hair cell-specific enhancer

Cochlear outer hair cells (OHCs) are essential for hearing. A short, OHC-specific enhancer is necessary but not yet available for gene therapeutic applications in OHC damage. Such damage is a major cause of deafness. Prestin is a motor protein exclusively expressed in OHCs. We hypothesized that the cis-regulatory DNA fragment deletion of Slc26a5 would affect its expression. We tested this hypothesis by conducting CRISPR/Cas9-mediated large DNA fragment deletion of mouse Slc26a5 intron regions. First, starting from a similar to 13 kbp fragment, step-by-step, we narrowed down the sequence to a 1.4 kbp segment. By deleting either a 13 kbp or 1.4 kbp fragment, we observed delayed Prestin expression. Second, we showed that 1.4 kbp was an OHC-specific enhancer because enhanced green fluorescent protein (EGFP) was highly and specifically expressed in OHCs in a transgenic mouse where Rill' was driven by the 1.4 kbp segment. More importantly, specific RIR' was also driven by its homologous 398 by fragment in human Slc26a5. This suggests that the enhancer is likely to be evolutionarily conserved across different species.

Automated summary

Cochlear outer hair cells (OHCs) are important for hearing, and a specific enhancer for OHC damage treatment is currently unavailable. This study used CRISPR/Cas9 to delete large DNA fragments of Slc26a5 in mice and found that the deletion affected the expression of Prestin. Furthermore, the study identified a 1.4 kbp segment as an OHC-specific enhancer, which is likely to be evolutionarily conserved across different species.